Two principal contact-dependent mechanisms are used by CTL or NK cells to kill their respective targets: engagement of death receptors of the TNF family, such as Fas, or via exocytosis of cytotoxic granules. The granule exocytosis pathway has been determined to be dominant in elimination of virus-infected cells, protection against other intracellular pathogens, and tumor surveillance. The exocytosed granules contain a cocktail of cytotoxic proteins including perforin and a family of serine proteases, granzymes. Granzyme A and granzyme B (GrB) are the most abundant granzymes, and GrB is the only granzyme to share substrate specificity with the caspase family. It cleaves its substrate after an aspartate residue, allowing it to mimic an initiator caspase in triggering target cell apoptosis. Despite GrB's ability to engage the death pathway at multiple entry points, recent evidence suggests a significant role for mitochondrial apoptotic events in its function. The overall goal of the current application is to elucidate novel apoptotic pathways utilized by GrB to mediate the mitochondrial apoptotic cascade. Our previous studies have elucidated a requirement for Bak, a mitochondrial resident and a proapoptotic Bcl-2 family member for a mitochondrial response toGrB. However, it is not clear what links GrB in the cytosol to mitochondrial Bak. Our preliminary data suggest that Mcl-1, an antiapoptotic Bcl-2 family member that resides on the mitochondrial outer membrane mediates crosstalk between the mitochondria and cytosolic GrB. We hypothesize that mitochondrial amplification of the caspase cascade is a significant component of GrB mediated apoptosis, and as such may serve as a target for regulation of GrB function, i.e. enhancing GrB activity against transformed cells and inhibiting its activity in graft rejection. To test this hypothesis, we propose to elucidate the functional mechanisms and the cascading nature of GrB mediated mitochondrial apoptosis. We propose to focus on three distinguishable phases of the cascade: (i) the role of Mcl-1 in mediating an upstream signal to initiate the mitochondrial cascade; (ii) the roles of Bax and Bak in the execution of the GrB-mediated cascade; and (iii) the role of XIAP as a GrB inhibitor that is hypothesized to function both upstream and downstream of the mitochondrial apoptotic loop. The proposed studies are expected to characterize unknown effector mechanisms used by cytotoxic lymphocytes to kill target cells, and to contribute to the development of strategies to prevent graft rejection and overcome apoptosis resistance in viral infected or transformed cells.